Study On Identification Of HCMV Coding Potential SmORF And Inhibition Of HCMV MCP Expression And Replication By EGS

Human cytomegalovirus(HCMV),belonging to the subfamily of ?-herpesvirus,is the major viral etiological factor of congenital birth defects and one of the most important pathogen capable of causing serious disease in immune-na?ve individuals and immunocompromised.HCMV has the largest genetic content among the human herpesvirus species with estimates of 515 small open reading frames(sm ORFs)by ribosome profiling and transcript analysis.However,limited available information is known about HCMV sm ORFs coding potential and the biological function of sm ORFs-encoded polypeptides(SEP)up to now.The present study focused on identification of HCMV coding sm ORF and exploration of its SEP biological function.In the present research,we screend out 10 SEP with immunogenicity by bioinformatics analysis and then produced antibodies against polypeptides;we validated that the anti-r ORF73 and anti-ORFL297 C.i ORF1 polypeptide antibody could specifically detect the SEP,respectively.Moreover,ORFL297 C.i ORF1 polypeptide was naturally,endogenously produced in cells infected with HCMV;and then we validated that ORFL297 C.i ORF1 encoding a polypeptide by transient transfection in cells.We dmonstrated for the first time that ORFL297 C.i ORF1 polypeptide significantly promoted the growth of HCMV;ORFL297C.i ORF1 polypeptide also decreased the expression of IFN-? and increased the expression of TGF-?1.What's more,ORFL297 C.i ORF1 polypeptide consequently downregulated the CEACAM1 and activated Smad3 of TGF-? signaling pathways,respectively.Our results indicated that ORFL297 C.i ORF1 polypeptide promoted the replication of HCMV by regulating TGF-? signaling pathways,and therefore protected against the host innate immune response.The present study indicated that the possibility of the protein-coding potential of HCMV sm ORF and its SEP might possess some biological functions.Therefore,identification of the coding sm ORF may contribute to understanding HCMV further and provide new insight into to the prevention and treatment of HCMV infection.Up to now,no available vaccine for preventing HCMV infection and the emergence of drug-resistant viral strains appeal to develop new drug and more effective treatment strategies.HCMV major capsid protein(MCP)plays an important role in the formation/maturation of viral capsid and growth of HCMV,suggesting that MCP may serve as a target to treat of HCMV infection.External guide sequences(EGSs),the short RNAs,guide ribonuclease P(RNase P)that is a t RNA processing enzyme to specifically degrade the targeting m RNA by forming a pre-t RNA-like complex;and therefore,the EGS technology based on RNase P may serve as a potential tool knocking down gene expression.We previously reported that EGS guided RNase P to cleave a target m RNA.In this study,HCMV MCP m RNA was selected as the target to design an EGS variant.In vitro assay of EGS directed RNase P to degradation of the HCMV MCP m RNA,the EGS variant MCP-V625 was about 80-fold more efficient than EGS MCP-SER derived from a natural pre-t RNA.Moreover,the inhibition level of MCP m RNA expression was 98% and 76% in the cell lines expressing EGS variant MCP-V625 and a natural pre-t RNA-derived EGS MCP-SER and a decrease of viral replication by about 10000-and 200-fold in cells with HCMV infection,respectively.A further study implied that inhibiting of the MCP expression by MCP-SER and MCP-V625 resulted in blocking the steps of both of the HCMV capsid maturation and formation but not affected HCMV genomic DNA replication,promoting the replication of HCMV.Our results indicated that the designed EGS variant MCP-V625 exhibited more efficient than EGS MCP-SER originated from the natural pre-t RNA in blocking HCMV genes expression and viral growth.Therefore,our findings imply that the designed EGS variant targeting to HCMV MCP m RNA may serve as a potent candidate agent to treat diseases caused by HCMV infection.